Steroid modulation of human serum albumin binding properties. A spin label study.

The binding isotherm and unique electron spin resonance spectral characteristics of a monoanionic spin label (1-gamma-aminobutyrate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene) and a dianionic spin label (1-glutamate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene) are used to prove the steroid modulation of serum albumin binding properties. Effects of a selected number of steroids (progesterone, testosterone, estradiol, aldosterone, estriol, corticosterone, deoxycorticosterone, hydrocortisone, and cortisone) on the binding isotherm of the monoanionic spin label binding to serum albumin have been determined. At the steroid/albumin ratio of 0.5 to 1, progesterone, testosterone, and estradiol enhance binding of the spin label at all concentrations studied. However, the remaining steroids exert an inhibitory effect at low spin label/albumin ratios and an enhancement effect at high spin label/albumin ratios. Progesterone and cortisone effects on the resonance spectra of the spin label bound to serum albumin confirm the enhancement and displacement properties of these ligands. Thus, like fatty acids, steroids may bind to either the primary or secondary bilirubin binding sites and also allosterically perturb the binding properties of serum albumin. The in vivo importance of the steroid-albumin interaction is discussed.     

Analysis of existing models of spin label movement during spin labeling of biological macromolecules

The spin-labeled bovine serum albumin and IgG were studied in search of an experimental approach for comparison of different models of rotational mobility of spin label. These models are: the model of isotropic motion of spin label together with the macromolecule (IM); the model of highly anisotropic motion of spin label (HAM); and the model of slow isotropic motion of label around the binding site (SIML). The experimental spectra were measured on a common X-band ESR spectrometer and on the unique 140 GHZ (lambda = 2 mm) ESR spectrometer under the same conditions. Theoretical spectra were computer-calculated according to Freed's theory. We have found, that the results of temperature-viscosity experiments in X-band are contradictory to the model of IM both for the BSA and IgG species. The models of HAM and SIML for the BSA give identical X-band spectra. The bovine serum albumin spectra in the 2 mm region strongly contradict to the assumptions of the HAM model. Also, the SIML model fails to describe the experimental spectra in terms of isotropic motion of the spin label around the binding site. X-band spectra of IgG can not be explained by the SIML model, while the same spectra in the 2 mm region can not be explained by the HAM model.     

Fatty acid enhancement of human serum albumin binding properties. A spin label study.

The introduction of a new spin-labeled anionic ligand, 1-gamma-aminobutyrate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene, is reported. Under the experimental conditions, the first molar equivalent of this ligand is 93% bound to human serum albumin. With the addition of palmitate, the free spin label concentration decreases greatly, by almost 80%, in the presence of a fatty acid:albumin ratio of 3:1 to 4:1. The spectral characteristics of the bound spin label are also affected. The changes seen in the intensity of and the splitting between the high and low field extrema are indicative of perturbations of the protein molecule. It is seen then that the binding of each molar equivalent of fatty acid effects the conformation state of albumin and allosterically affects albumin binding properties. Computer spectral subtractions, furthermore, suggest that the binding of the first molar equivalent of palmitate specifically increases the affinity of the first two 1-gamma-amino-butyrate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene binding sites. The present results indicate that fluctuations in serum free fatty acid levels within the physiological range may have a major modulatory effect on the free serum levels of certain drugs and/or physiological substances that bind to albumin.     

Location of long chain fatty acid-binding sites of bovine serum albumin by affinity labeling

Affinity labeling with palmitic acid was used to identify long chain fatty acid-binding sites of bovine serum albumin. [1-14C]Palmitic acid was activated by esterification with N-ethyl-5-phenyl-isoxazolium-3'-sulfonate (Woodward's Reagent K). The product was purified by chromatography and shown to compete with unesterified fatty acids for binding sites on bovine serum albumin. Activated [14C]palmitic acid coupled covalently to albumin producing [14C]palmitoyl-albumins containing from 0.12 to a maximum of 6.9 mol of attached label per mol of albumin. The presence of the covalently attached affinity label depressed binding of other long chain fatty acids to albumin. Albumin carrying 1 eq. of [14C]palmitate was cleaved using cyanogen bromide, pepsin, and trypsin. Radioactive peptides were isolated by high pressure liquid chromatography. Three peptides accounted for greater than 90% of the label. Residues labeled with [14C]palmitate were identified as Lys-116, Lys-349 and Lys-473, and the relative distribution of label was 10, 45, and 45% respectively, consistent with the presence of two strong binding sites in the COOH-terminal half of albumin and a somewhat weaker site in the NH2-terminal half.     

Spin label study of slow molecular rotations of globular proteins using microwave saturation effects in electron paramagnetic resonance

Viscosity, temperature and ionic strength dependences of ESR microwave saturation parameters of spin labelled human oxyhemoglobin (Hb) and bovine serum albumin (BSA) have been studied. The piperidine and pyrrolidine nitroxyl derivatives of maleimide were used as covalent SH reagents for Hb and BSA and the same two derivatives of gamma-benzocarboline and spin labelled stearic acid were used as noncovalent spin probes for BSA. The effects of label binding tightness on ESR spectral parameters were considered. The rotational correlation times were determined using viscosity dependences of the separation of the outer hyperfine extrema and Stokes extrapolations at high viscosities. The ESR microwave saturation parameters of the spin labels were shown to depend just weakly on temperature (at constant eta/t) over the range 0-25 degrees and on g, A values but to be sensitive to protein rotational correlation times up to 10(-4) sec and also to the rotational anisotropy and to the relative motion of the spin label.     

Thiol-reactive clenbuterol analogues conjugated to bovine serum albumin

Several novel thiol-reactive clenbuterol analogues were coupled in high yield with bovine serum albumin (BSA). After labelling of unreacted cysteines with maleimide spin label (MiSL), the yield of the coupling reaction was determined by electron paramagnetic resonance (EPR) spectroscopy and spectral analysis. Two spin-probe populations with different mobility states were quantitatively determined. Molecular dynamics was used to model the structure of clenbuterol analogues and spin label conjugated to BSA and recognition of conjugates by anti-clenbuterol antibodies was demonstrated. The recognition of BSA-A, BSA-C and BSA-S conjugates with monoclonal and polyclonal anti-clenbuterol (mCLB-Ab and rCLB-Ab) antibodies was an indication, that chlorine substituents on the aromatic ring of clenbuterol derivatives are not necessary for the binding of antibodies to the conjugates. These results confirmed the importance of the tert-butylamino group as a part of the epitope and contribute to the understanding of the recognition process with anti-clenbuterol antibodies.     

Characterization of protein spin labeling by maleimide: evidence for nitroxide reduction

A quantitative determination of maleimide spin label (MAL) binding in oxi and met hemoglobin (Hb) and bovine serum albumin are investigated using double integration to the ESR signal. This determination permitted the observation that a considerable fraction of MAL is reduced, losing its paramagnetism. Experiments using the same spin label with myoglobin and Hb with blocked-SH groups, where reduction was not observed, indicate the involvement of SH groups in the process. The 4-hydroxy-2,2,6,6-tetramethylpiperidino-1-oxyl spin label (which is not able to bind in the SH group) is reduced too, but the dependence on the molar ratio is different in comparison with the MAL case. In both cases the reduction percentage depends on the molar ratio spin label to protein and to the protein concentration. In order to obtain the total SH groups labeled (two in the Hb case) it is necessary to use an excessive amount of label (around 18:1) in the 0.5 mM Hb concentration.     

Study of steroid-proteininteractions by electron spin resonance spectroscopy. Binding of a spin-labelled dihydrotestosterone to bovine serum albumin.

The interaction of bovine serum albumin with dihydrotestosterone bearing a spin label at C-3 was studied using electron spin resonance (ESR) spectroscopy. Quantitative binding parameters (Ka approximately 10(5) M-1; maximum binding capacity; two sites/mol albumin) obtained by ESR were in good agreement with those given by equilibrium dialysis. ESR study at various temperatures allowed the calculation of the thermodynamic parameters of the steroid-protein interaction: deltaG=-6.8 kcal/mol; deltaH=-7.9 kcal/mol; deltaS=-3.2 cal/mol per degree and confirmed a transition temperature of about 65 degrees C for albumin. Na, Liland Ca salts had a generally favorable effect on the interaction whereas other ions (e.g. Hg, Cu) impaired the binding process. Study of the width of the ESR spectra of the protein-bound spin-labelled steroid and extrapolation of a 2 T value to infinite viscosity (Azz coupling constant) indicated a non-polar binding site, which became increasingly hydrophobic as the temperature was raised. Since this methodology can give both pertinent quantitative and qualitative data, ESR spectroscopy should be of value in the study of steroid-protein interactions of biological significance.     

Affinity labeling of the primary bilirubin binding site of human serum albumin.

A label for the bilirubin binding sites of human serum albumin was synthesized by reacting 2 mol of Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate) with 1 mol of bilirubin. This yielded a water-soluble derivative in which both carboxyl groups of bilirubin were converted to reactive enol esters. Covalent labeling was achieved by reacting the label with human serum albumin under nitrogen at pH 9.4 and 20 degrees. Under the same conditions, no covalent binding to the monomers of several proteins could be demonstrated. The number of binding sites for bilirubin and the label were found to be the same, and competition experiments with bilirubin showed inhibition of covalent labeling. The absorption, fluorescence and CD spectra of the label in a complex with human serum albumin were similar to those of the bilirubin human serum albumin complex. However, following covalent attachment to the spectral properties were changed, indicating loss of conformational freedom of the chromophore. Labeling ratios were selected to result in the incorporation of less than 1 mol of label/mol of human serum albumin. Under these conditions, labeling is thought to occur primarily at the high affinity binding site.     

Intra-albumin migration of bound fatty acid probed by spin label ESR

Conventional ESR spectra of 16-doxyl-stearic acid bound to bovine and human serum albumin were recorded at different temperatures in order to investigate the status of spin-labeled fatty acid in the interior of the protein globule. A computer spectrum simulation of measured spectra, performed by non-linear least-squares fits, clearly showed two components corresponding to strongly and weakly immobilized fatty acid molecules. The two-component model was verified on spectra measured at different pH. Thermodynamic parameters of the spin probe exchange between two spin probe states were analyzed. It was concluded that at physiological conditions, fatty acid molecules permanently migrate in the globule interior between the specific binding sites and a space among albumin domains.     

A spin label study of the thyroid hormone-binding sites in human plasma thyroxine transport proteins

The binding site topographies of the three thyroid hormone-transporting proteins in human serum--prealbumin, thyroxine binding globulin, human serum albumin--have been studied with the aid of five spin-labeled analogs of L-thyroxine in which the distance between the phenolic hydroxyl and the nitroxide nitrogen ranged from 17 to 23 A. In the presence of prealbumin, the electron spin resonance spectrum of 3-([alpha-carboxy-4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenethyl]-carbamoyl)-2,2,5,5-tetramethyl-3-pyrrolinl-yloxy-ethyl ester revealed the presence of a highly immobilized spin label. As the chain length between the thyroxyl moiety and the pyrroline ring was increased, the mobility of the nitroxide group in the prealbumin-bound labels increased. If the spin labels bind in an extended conformation, the thyroxine-binding site was estimated to be approximately 21 A in depth. This finding is consistent with the known crystal structure of prealbumin and suggests that the solution and crystal conformations of the protein are very similar. In contrast to prealbumin, the thyroxine-binding site on thyroxine-binding globulin was found to be more open and possibly deeper. Human serum albumin has two binding sites for thyroxine, one of which has a higher affinity and is deep enough to accommodate a molecule that is 23 A in length. The lower affinity site is somewhat shallower and probably wider, as thyroxine spin labels bound to this site exhibited greater mobility.     

Studies on the mechanism of inhibition of nuclear triiodothyronine binding by fatty acids

Studies were designed to elucidate the mechanism by which unsaturated fatty acids inhibit the binding of triiodothyronine (T3) ro rat liver nuclei. The possibility of a direct interaction between oleic acid and T3 was excluded by dialysis experiments. Oleic acid inhibits nuclear T3 binding in a strictly competitive manner. The Ki value of oleic acid was approx. 10(4) times greater than that of T3. The inhibitory effect of oleic acid could be reversed by bovine serum albumin.     

Electron paramagnetic resonance study of the interactions between steroid hormones and binding proteins]

Interaction of a spin labeled corticosteroid (desoxycorticosterone nitroxyde: DOC -NO) with three purified proteins (albumin, transcortin, progesterone binding protein: PBG) was studied by electron spin resonance (ESR) spectroscopy. DOC-NO was competitive with natural corticosteroids and therefore bound at the same site to specific binding proteins. ESR spectra in the presence of each of the proteins showed an immobilized (bound) form of the spin labeled steroid and allowed the calculation of the corresponding association constant (Ka) at equilibrium. The three binding proteins could be characterized by the ESR parameters of the DOC-NO bound form. The thermodynamic parameters (deltaH, deltaS) of the steroid-protein interactions were calculated from the ESR data obtained within a wide temperature range (3--40 degrees C). The ESR spectra width (2T) was used to evaluate the polarity of the spin label environment within the steroid binding site: a hydrophobic character was observed for transcortin whereas PBG exhibited a more hydrophilic steroid binding sits. The rotational correlation time of the three protein DOC-NO complexes at equilibrium were calculated from ESR data; the results were correlated with the protein molecular size and suggested a non spherical shape for the binding macromolecule in solution. Spin labelling of biologically active steroids thus provides a novel approach for the study of the interaction of these hormones with their binding protein. Providing a suitable spin label, the ESR parameters may allow the characterization of several types of binding sites of different biological significance for the same hormone, in biological fluids as well as in target tissues.     

Spin-Labeled Neurospora Mitochondria

Spin-label studies were carried out on Neurospora mitochondria under in vivo and in vitro labeling conditions. A long-chained spin-labeled fatty acid was incorporated by Neurospora and was found in mitochondrial phospholipids. The molecular motion at various temperatures was different from that for the same spin label under in vitro labeling conditions. The results for spin-labeled mitochondria were compared with those from isolated lipids and with those from aggregates of spin-labeled fatty acid and isolated bovine serum albumin. These comparisons suggest that the hydrocarbon portions of membranes are relatively fluid and are not extensively restricted in motion by association with proteins.     

Radiochemical synthesis and photochemical properties of the uncoupler 2-azido-4-nitrophenol, a versatile photoaffinity labeling reagent.

2-Amino-4-nitrophenol was tritiated in an acid-catalyzed hydrogen exchange reaction. Radioactive 2-azido-4-nitrophenol with a specific radioactivity up to 21 mCi/mmol was synthesized from 2-amino-4-nitrophenol by diazotization and azide coupling. The photochemical properties of the uncoupler, 2-azido-4-nitrophenol, were studied as free solute and as ligand bound to uncoupler binding sites in bovine serum albumin and mitochondria. Based on product analyses, irradiation of free or bound 2-azido-4-nitrophenolate with visible light results in the formation of nitrene intermediates with a singlet to triplet ratio of 6:1 to 9:1. 2-Azido-4-nitrophenolate and bovine serum albumin form a strong 1:1 complex (KD = 0.7 micron) which can be converted into a photoproduct with a covalent bond between the label and the protein. The acid dissociation constant of the protein-bound 2-amino-4-nitrophenol moiety is strongly pH dependent. Photoaffinity labeling of mitochondria by 2-azido-4-nitrophenolate follows a pattern expected from equilibrium binding studies using normal and lipid-depleted particles: polypeptides were found to bear 90-95% of the radioactive label, and 5-10% of the latter was bound to phospholipids. Two polypeptides (approximately 56 000 and 31 000 daltons) were associated with 60% of the label, indicating a high degree of specific photochemical labeling.     

A spin label study of horseradish peroxidase.

The topography of the active sites of native horseradish peroxidase and manganic horseradish peroxidase has been studied with the aid of a spin-labeled analog of benzhydroxamic acid (N-(1-oxyl-2,2,5,5-tetramethylpyrroline-3-carboxy)-p-aminobenzhydroxamic acid). The optical spectra of complexes between the spin-labeled analog of benzhydroxamic acid and Fe3+ or Mn3+ horseradish peroxidase resembled the spectra of the corresponding enzyme complexes with benzhydroxamic acid. Electron spin resonance (ESR) measurement indicated that at pH 7 the nitroxide moiety of the spin-labeled analog of benzhydroxamic acid became strongly immobilized when this label bound to either ferric or manganic horseradish peroxidase. The titration of horseradish peroxidase with the spin-labeled analog of benzhydroxamic acid revealed a single binding site with association constant Ka approximately 4.7 . 10(5) M-1. Since the interaction of ligands (e.g. F-, CN-) and H2O2 with horseradish peroxidase was found to displace the spin label, it was concluded that the spin label did not indeed bind to the active site of horseradish peroxidase. At alkaline pH values, the high spin iron of native horseradish peroxidase is converted to the low spin form and the binding of the spin-labeled analog of benzhydroxamic acid to horseradish peroxidase is completely inhibited. From the changes in the concentration of both bound and free spin label with pH, the pK value of the acid-alkali transition of horseradish peroxidase was found to be 10.5. The 2Tm value of the bound spin label varied inversely with temperature, reaching a value of 68.25 G at 0 degree C and 46.5 G at 52 degrees C. The dipolar interaction between the iron atom and the free radical accounted for a 12% decrease in the ESR signal intensity of the spin label bound to horseradish peroxidase. From this finding, the minimum distance between the iron atom and nitroxide group and hence a lower limit to the depth of the heme pocket of horseradish peroxidase was estimated to be 22 A.     

Long chain fatty acid binding to human plasma albumin.

The binding of six physiologically important long chain fatty acids to defatted human plasma albumin was measured at 37 degrees in a calcium-free Krebs-Ringer phosphate buffer, pH 7.4. The data were analyzed in terms of multiple stepwise equilibria. With the saturated acids, the magnitude of the equilibrium (association) constants, Ki, increased as the chain length increased: laurate smaller than myristate smaller than palmitate smaller than stearate. Oleate was bound more tightly than stearate; by contrast, linoleate was bound less tightly than stearate. The equilibrium constants, K1 through K12, ranged from 2.4 times 10-6 - 3.5 times 10-3 m-1 for laurate to 2.6 times 10-8 - 3.5 times 10-5 m-1 for oleate. Successive values of Ki decrease for each of the acids, indicating that major cooperative binding effects do not occur over the physiological range of fatty acid concentrations. In no case could the Ki be segregated into distinct classes, suggesting that any grouping of albumin binding sites is somewhat arbitrary. The results were inconclusive concerning whether premicellar association of unbound fatty acid occurs. Although corrections for premicellar association produced very little change in the Ki values for myristate, they raised the Ki for palmitate and stearate by 300 to 700 per cent. A sigmoidal relationship was obtained when the logarithm of Ki was plotted against chain length for the saturated fatty acids containing 6 to 18 carbon atoms, indicating that the binding energy is not simply a statistical process dependent only on the fatty acid chain length. This selectivity that albumin contributes to the binding process may be due to varying degrees of configurational adaptability of its binding sites as the fatty acid increases in length.     

ESR spectral changes induced by chlorpromazine in spin-labeled erythrocyte ghost membranes

chlorpromazine interacted preferentially with membrane proteins rather than membrane lipids in the initial incorporation into human erythrocyte ghosts, as demonstrated by means of the fluorescence quenching and a maleimide spin label. In this state the membrane fluidity increased. At higher concentrations of chlorpromazine, the membrane fluidity decreased and a motionally restricted signal from fatty acid spin labels appeared predominantly. However, no such signal appeared in protein-free vesicles. The temperature and pH dependences of the outer hyperfine splitting of this restricted signal were very similar to those of bovine serum albumin. On the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of chlorpromazine-treated and -untreated ghosts, it was found that there was no significant difference in membrane proteins between both samples except for the changes of a few bands which were not directly concerned with the occurrence of this restricted signal. These results suggest that the fatty acid spin labels bind preferably to membrane proteins as the lipid domain becomes packed with chlorpromazine.